Overview of Bone Grafts
The rapid and effective repair of bone defects caused by injury, disease, wounds, or surgery has long been a goal of orthopaedic surgery. Bone grafting is a well established surgical technique. Sources of bone are autograft (primarily from cancellous bone sources), allograft (generally comprising cancellous bone and structural cortical pieces), and xenograft (typically cancellous bone). With any bone graft, it is advantageous for the graft to integrate quickly with the host bone and then to be remodeled into host bone. In structurally loaded graft sites, it is desired that the bone graft integrate while maintaining its strength throughout the remodeling process.
Several compositions and materials have thus been used or proposed for use in the repair of bone defects. The biological, physical, and mechanical properties of the compositions and materials are among the major factors influencing their suitability and performance in various orthopaedic applications. Desirably, materials used for the repair of bone defects are remodeled—the material being resorbed and replaced by similar host tissue. For example, implanted bone being replaced by host bone.
Bone, both cortical and cancellous, has been used in the repair of bone defects. As will be discussed, bone remodeling, including resorption of the implanted bone material and formation of new bone material, is desirable for implanted bone material. Reference is thus made to resorption rates as a guide to rates of remodeling. Cortical bone is stronger than cancellous bone but is not resorbed or remodeled as quickly as cancellous bone. Complete remodeling of cortical bone may take ten or more years. Consequently, many surgeons prefer cancellous bone for bone grafting. However, because cancellous bone does not have the strength of cortical bone, it is not suitable for all applications.
Cortical bone comprises approximately 70% mineral, 20% protein (primarily Type 1 structural collagen), and 10% water. The mineral comprises very small (nanoscale) crystals of impure hydroxyapatite. These crystals have a large surface area and are reasonably resorbable. However, in cortical bone, the collagen structure is dense and acts as a limiting factor in resorption. The resorption rate of the collagen structure is limited by the fact that initial degradation occurs only by the action of the specific enzyme collagenase.
Resorption of cancellous bone is generally faster than resorption of cortical bone. Among other things, pores in the cancellous bone allow cells to infiltrate and grow new bone, while providing a large surface area for enzymatic attack to occur on the collagen.
Much effort has been invested in the identification and development of bone graft materials, including treating bone for such use. Urist has published seminal articles on the theory of bone induction and a method for decalcifying bone, i.e., making demineralized bone matrix (DBM). Urist M. R., Bone Formation by Autoinduction, Science 1965; 150(698):893-9; Urist M. R. et al., The Bone Induction Principle, Clin. Orthop. Rel. Res. 53:243-283, 1967. DBM is an osteoinductive material, in that it induces bone growth when implanted in an ectopic site of a rodent, owing to the osteoinductive factors contained within the DBM.
DBM implants have been reported to be particularly useful (see, for example, U.S. Pat. Nos. 4,394,370, 4,440,750, 4,485,097, 4,678,470, and 4,743,259; Mulliken et al., Calcif Tissue Int. 33:71, 1981; Neigel et al., Opthal. Plast. Reconstr. Surg. 12:108, 1996; Whiteman et al., J. Hand. Surg. 18B:487, 1993; Xiaobo et al., Clin. Orthop. 293:360, 1993, each of which is incorporated herein by reference). DBM typically is derived from cadavers. The bone is removed aseptically and treated to kill any infectious agents. The bone may be particulated by milling or grinding, and then the mineral component is extracted by various methods, such as by soaking the bone in an acidic solution. The remaining matrix is malleable and can be further processed and/or formed and shaped for implantation into a particular site in the recipient. Demineralized bone prepared in this manner contains a variety of components, including proteins, glycoproteins, growth factors, and proteoglycans. Following implantation, the presence of DBM induces cellular recruitment to the site of injury. The recruited cells may eventually differentiate into bone forming cells. Such recruitment of cells leads to an increase in the rate of wound healing and, therefore, to faster recovery for the patient.
Many of the processes used to prepare tissue for transplant cause some collagen damage. These processes include, for example, treatment with oxidizing agents such as peroxides, irradiation, and autoclaving. While limited collagen damage to the tissue may increase the rate of bone remodeling, too much collagen damage (as often occurs from such treatments) leads to replacement of the tissue with undesirable fibrous tissue.
Overview of Collagen
Collagen is the major component of extracellular matrix (ECM) of many tissues including bone, tendon, ligament, skin and others. Collagen is organized in fibrillar bundles. In tissue, the organization of collagen matrix is essential for the mechanical properties. In addition, the oriented fibrillar structure of collagen facilitates cellular recognition and provides a suitable carrier for many biological active molecules such as growth factors including BMPs. It has been demonstrated to be important for cell attachment, proliferation, differentiation, and remodeling or reorganization. In processing of tissue grafting materials, in some specific applications, it may be desirable that the natural collagen structures are preserved.
Overview of Bone Sterilization and Bone Remodeling
It is generally desirable that bone grafts be free of disease causing pathogens such as viruses, bacteria, mold, fungus, and yeast. Viruses are a specific type of pathogen. Viruses are active inside cells but not in the general environment. If viruses are present in bone graft material, then they were present in the tissue before harvest. Once viruses are inactivated, it is unlikely that the tissue will become recontaminated with viruses. This is in contrast to bacteria, mold, etc., which can readily recontaminate tissue unless special precautions are taken to surround the tissue with a sterile barrier or process it in a sterile environment.
To ensure that the tissue is free of pathogens, the tissue is typically screened for possible diseases, may be processed aseptically, and additional cleaning/disinfecting steps may be carried out. Pathogen inactivation or removal depends on various factors including temperature, pressure, time, and the use of chemical agents. Collagen damage may result from pathogen inactivation processes. Examples of collagen damaging sterilization/viral inactivation techniques include treatment with harsh oxidizing agents, radiation, or autoclaving. Other pathogen inactivation processes, such as detergent or alcohol rinses, cause little or no collagen damage.
Supercritical, critical or near critical fluids have been used to remove or inactivate virus or virus-like particles (U.S. Pat. No. 5,877,005; U.S. Pat. No. 6,217,614 B1; U.S. Pat. No. 7,008,591; White et al., J. Biotech. 123:504, 2006). These methods generally apply supercritical fluids with other chemical agents, or apply supercritical fluids at relatively low temperature such as below 60° C., or apply supercritical fluids to a solution of a biological material. Treatment with supercritical fluids at lower temperature does not always inactivate all pathogens, especially non-enveloped viruses. On the other hand, the use of chemical agents may destroy the biological activity of the materials such as bone grafting materials.
Bone remodeling is a dynamic process by which old bone is removed from the skeleton and new bone is added. Bone remodeling comprises two stages: resorption and formation. One method of improving bone remodeling is to degrade collagen to facilitate the resorption stage of bone remodeling.
Accordingly, pathogen inactivation processes that cause collagen damage may increase the rate of bone resorption. This may not, however, lead to bone remodeling. The collagen damage sometimes can result in the bone being replaced by undesirable fibrous tissue instead of bone. Bone that has been subjected to harsh treatments, such as autoclaving or high radiation doses, to sterilize the bone often resorbs quickly but is not replaced by host bone. These harsh treatments break down collagen in the bone but do so in a way that the implanted bone often causes chronic inflammation—the implanted bone having been replaced by fibrous tissue. For this reason, sterilization/viral inactivation treatments that damage collagen are generally limited in their time or harshness (low peroxide concentrations, low radiation doses, etc.) in order to reduce collagen damage. While such limiting does reduce collagen damage, it also compromises the effectiveness of the treatments.
Thermal treatment of bone, for example by autoclaving or using dry heat, for sterilization is not typically done. Bone that has been sterilized by these techniques is generally found to be resorbed but not remodeled. Thus, while heating is simple, rapid, and leaves no chemical residues, the lack of remodeling following implantation of graft material has made it largely undesirable.
It would be useful to have a method of sterilization without substantially degrading biological properties of the bone.